Recombinant mycobacterium as an immunotherapeutic agent for the treatment of cancer

ABSTRACT

The invention relates to a recombinant  Mycobacterium  cell for use as an immunotherapeutic agent in the treatment of cancer, particularly in the treatment of solid tumors. More particularly, the invention relates to the immunotherapy of bladder carcinoma.

This application is a divisional of U.S. Ser. No. 15/571,415 filed Nov. 2, 2017, now U.S. Pat. No. 10,525,118, which is a 35 U.S.C. 371 National Phase Entry Application from PCT/EP2016/059872, filed May 3, 2016, which claims the benefit of European Patent Application No. 15166206.1 filed on May 4, 2015 and U.S. Ser. No. 62/387,407, filed Dec. 23, 2015, the disclosures of which are incorporated herein in their entirety by reference.

The invention relates to a recombinant Mycobacterium cell for use as an immunotherapeutic agent in the treatment of cancer, particularly in the treatment of solid tumors. More particularly, the invention relates to the immunotherapy of bladder carcinoma.

Urothelial bladder carcinoma is the 5th most common cancer. In the United States, about 75.000 new cases are diagnosed each year 4.5% of all new cancers, and approximately 15.600 deaths are expected. In Germany, about 16.000 new cases are diagnosed each year. Because a recurrence of disease is likely in bladder carcinoma, patients must undergo surveillance for an extended period.

Most bladder carcinomas begin in transitional epithelial cells that make up the inner lining of the bladder. As these tumors grow, they can invade the surrounding connective tissue and muscle. In advanced disease, tumors spread beyond the bladder to nearby lymph nodes or pelvic organs or metastasize to more distant organs such as lung, liver and bone.

The overall 5-year survival rate for bladder carcinoma is 77%, and this rate has not changed significantly over the last 10 years. When considered by stage, the 5-year relative survival rates for patients with tumors restricted to the inner layer of the bladder are 96% and 69%, respectively. The rates drop to 34% for those with disease that has spread locally beyond the bladder and to 6% with distant metastases.

For patients with non-muscle invasive bladder carcinoma, treatment usually involves a surgical removal of the tumor followed by chemotherapy, usually mitomycin C, within the bladder (so-called intravesical chemotherapy). After recovering from surgery, patients with a lower risk of disease progression may undergo surveillance or additional intravesical chemotherapy. Patients with moderate-to-high-grade disease often receive intravesical immunotherapy with an attenuated live bacterium Bacillus Calmette Guérin (BCG). BCG was the first FDA-approved immunotherapy and helps reduce the risk of bladder carcinoma recurrence by stimulating an immune response that targets the bacteria as well as any bladder carcinoma cells. In some patients, however, BCG therapy has found to be less effective, particularly after repeated administration.

Standard treatment for patients with muscle-invasive bladder carcinoma includes cisplatin-based chemotherapy followed by surgical removal of the bladder or radiation therapy and concomitant chemotherapy. Recurrent bladder carcinoma may be treated with combination therapy regimens, including gemcitabine plus cisplatin or methotrexate, vinblastine, doxorubicin plus cisplatin.

In the treatment of bladder carcinoma, tumor recurrence is a major concern, even for patients with low-grade disease and requires extensive follow-up. Better treatments, such as novel immunotherapies, might reduce recurrence rates and improve the survival of patients with bladder carcinoma.

A recombinant BCG strain expressing a phagolysosomal escape domain is described in WO 99/10496, the content of which is herein incorporated by reference. The phagolysosomal escape domain enables the strain to escape from the phagosome of infected host cells by perforating the membrane of the phagosome. In order to provide an acidic phagosomal pH for optimal phagolysosomal escape activity, a urease-deficient recombinant strain was developed. This strain is disclosed in WO 2004/094469, the content of which is herein incorporated.

WO 2012/085101, the content of which is herein incorporated, discloses that a recombinant BCG strain expressing membrane-perforating listeriolysin (Hly) of Listeria monocytogenes and devoid of urease C induces superior protection against aerogenic challenge with Mycobacterium tuberculosis (Mtb) as compared to parental BCG in a preclinical model. Further, it is shown that both the recombinant and the parental strain induce marked Th1 immune responses, whilst only the recombinant BCG strain elicits are profound Th17 response in addition.

In the present study, it was found that a recombinant urease-deficient and listeriolysin-expressing recombinant BCG strain induces a superior immune response compared to the parenteral BCG in an animal model.

A subject-matter of the present invention is a recombinant Mycobacterium cell which comprises a recombinant nucleic acid molecule encoding a fusion polypeptide comprising:

(a) a domain capable of eliciting an immune response, and

(b) a phagolysosomal escape domain.

for use as an immunotherapeutic agent in the treatment of solid tumors.

A further aspect of the present invention is a method for the immunotherapeutic treatment of solid tumors in a subject in need thereof, comprising administering to said subject a recombinant Mycobacterium cell which comprises a recombinant nucleic acid molecule encoding a fusion polypeptide comprising:

(a) a domain capable of eliciting an immune response, and

(b) a phagolysosomal escape domain.

According to the present invention it was found that vesicular instillation of a recombinant BCG cell into the bladder of rats surprisingly results in an increased infiltration of urinary bladder tissue by lymphocytes, particularly CD4- and CD8-positive lymphocytes resulting in a high incidence of focal and/or multifocal lymphocytic infiltration. In contrast thereto, the urinary bladder tissue of animals treated with standard BCG showed CD4- and CD8-positive lymphocytes only as single cell infiltrate levels (diffuse infiltration). Further, administration of a recombinant BCG cell did not raise any safety issues.

Presently, a phase I/II clinical trial assessing safety and efficacy of intravesical instillation of recombinant BCG in human patients with recurrent non-muscle invasive bladder carcinoma after standard BCG therapy is conducted.

Thus, the present invention also relates to a recombinant Mycobacterium cell as described above for use as an immunotherapeutic agent in the treatment of solid tumors in order to obtain focal and/or multifocal lymphocytic infiltration, e.g. with CD4 and CD8 T cells at the site of administration. The recombinant Mycobacterium cell of the present invention is particularly suitable for use in human medicine.

The immunotherapeutic agent is a live recombinant Mycobacterium cell which comprises a recombinant nucleic acid molecule encoding a fusion polypeptide comprising (a) a domain capable of eliciting an immune response and (b) a phagolysosomal escape domain. The domain capable of eliciting an immune response is preferably an immunogenic peptide or polypeptide from a pathogen or an immunogenic fragment thereof.

The Mycobacterium cell is preferably an M. bovis cell, an M. tuberculosis cell, particularly an attenuated M. tuberculosis cell or other Mycobacteria, e.g. M. microti, M. smegmatis, M. canettii, M. marinum or M. fortuitum. More preferably, the cell is an attenuated recombinant M. bovis (BCG) cell, particularly an M. bovis BCG cell, more particularly a recombinant M. bovis BCG cell from strain Danish subtype Prague (Brosch et al., Proc. Natl. Acad. Sci. USA, 104 (2007), 5396-5601). In an especially preferred embodiment, the Mycobacterium cell is recombinant urease-deficient. In an especially preferred embodiment the ureC sequence of the Mycobacterium cell is inactivated (ΔUrec), e.g. by constructing a suicide vector containing a ureC gene disrupted by a selection marker gene, e.g. the hygromycin gene, transforming the target cell with the vector and screening for selection marker-positive cells having a urease negative phenotype. In an even more preferred embodiment, the selection marker gene, i.e. the hygromycin gene, is subsequently inactivated. In this embodiment, the cell is a selection marker-free recombinant Mycobacterium cell. Most preferably, the cell is selection marker-free recombinant BCG strain Danish subtype Prague characterized as recombinant BCG ΔUrec::Hly+.

The domain capable of eliciting an immune response is preferably selected from immunogenic peptides or polypeptides from M. bovis, M. tuberculosis or M. leprae or from immunogenic fragments thereof having a length of at least 6, preferably at least 8 amino acids, more preferably at least 9 amino acids and e.g. up to 20 amino acids. Specific examples for suitable antigens are Ag85B (p30) from M. tuberculosis, Ag85B (α-antigen) from M. bovis BCG, Ag85A from M. tuberculosis and ESAT-6 from M. tuberculosis and fragments thereof. In other embodiments, the domain capable of eliciting an immune response is selected from non-Mycobacterium polypeptides.

More preferably, the immunogenic domain is derived from the antigen Ag85B. Most preferably, the immunogenic domain comprises the sequence from aa.41 to aa.51 in SEQ ID No.2.

The recombinant nucleic acid molecule further comprises a phagolysosomal escape domain, i.e. a polypeptide domain which provides for an escape of the fusion polypeptide from the phagolysosome into the cytosol of mammalian cells. Preferably, the phagolysosomal escape domain is a Listeria phagolysosomal escape domain, which is described in U.S. Pat. No. 5,733,151, herein incorporated by reference. More preferably, the phagolysosomal escape domain is derived from the listeriolysin gene (Hly) of L. monocytogenes. Most preferably, the phagolysosomal domain is encoded by a nucleic acid molecule selected from: (a) a nucleotide sequence comprising nucleotides 211-1722 as shown in SEQ ID No.1, (b) a nucleotide sequence which encodes the same amino acid sequence as the sequence from (a), and (c) a nucleotide sequence hybridizing under stringent conditions with the sequence from (a) or (b).

Apart from the nucleotide sequence depicted in SEQ ID No.1 the present invention also comprises nucleic acid sequences hybridizing therewith. In the present invention the term “hybridization” is used as defined in Sambrook et al. (Molecular Cloning. A laboratory manual, Cold Spring Harbor Laboratory Press (1989), 1.101-1.104). In accordance with the present invention the term “hybridization” is used if a positive hybridization signal can still be observed after washing for one hour with 1×SSC and 0.1% SDS at 55° C., preferably at 62° C. and more preferably at 68° C., particularly for 1 hour in 0.2×SSC and 0.1% SDS at 55° C., preferably at 62° C. and more preferably at 68° C. A sequence hybridizing with a nucleotide sequence as per SEQ ID No.1 under such washing conditions is a phagolysosomal escape domain encoding nucleotide sequence preferred by the subject invention.

A nucleotide sequence encoding a phagolysosomal escape domain as described above may be directly obtained from a Listeria organism or from any recombinant source e.g. a recombinant E. coli cell containing the corresponding Listeria nucleic acid molecule or a variant thereof as described above.

Preferably, the recombinant nucleic acid molecule encoding for a fusion polypeptide contains a signal peptide encoding sequence. More preferably, the signal sequence is a signal sequence active in Mycobacteria, preferably in M. bovis, e.g. a native M. bovis signal sequence. A preferred example of a suitable signal sequence is the nucleotide sequence coding for the Ag85B signal peptide which is depicted in SEQ ID No.1 from nucleotide 1 to 120.

Further, it is preferred that a peptide linker be provided between the immunogenic domain and the phagolysosomal escape domain. Preferably, said peptide linker has a length of from 5 to 50 amino acids. More preferably, a sequence encoding a linker as shown in SEQ ID No.1 from nucleotide 154 to 210 or a sequence corresponding thereto as regards the degeneration of the genetic code.

The nucleic acid may be located on a recombinant vector. Preferably, the recombinant vector is a prokaryotic vector, i.e. a vector containing elements for replication or/and genomic integration in prokaryotic cells. Preferably, the recombinant vector carries the nucleic acid molecule of the present invention operatively linked with an expression control sequence. The expression control sequence is preferably an expression control sequence active in Mycobacteria, particularly in M. bovis. The vector can be an extrachromosomal vector or a vector suitable for integration into the chromosome. Examples of such vectors are known to the man skilled in the art and, for instance, given in Sambrook et al. supra.

The immunotherapeutic agent of the present invention is suitable for the treatment of solid tumors, such as bladder, lung, liver, breast, kidney or prostate tumors. Particularly, the present invention is suitable for the treatment of non-invasive solid tumors. In an especially preferred embodiment, the solid tumor is bladder carcinoma, e.g., non-invasive bladder carcinoma, e.g. non-invasive papillary carcinoma in situ (T_(a)), non-invasive carcinoma in situ (T_(cis)), tumor invading subepithelial connective tissue (T₁), tumor invading superficial muscle (inner half) (T_(2a)), tumor invading deep muscle (outer half) (T_(2b)), tumor invading perivesical tissue (T₃ including T_(3a) and T_(3b)), tumor invading prostate, uterus or vagina (T_(4a)), and tumor invading pelvic wall or abdominal wall (T_(4b)). Particularly, the tumor is a superficial tumor or carcinoma in situ (T_(cis)), non-invasive papillary carcinoma (T_(a)), or a tumor invading subepithelial connective tissue (T₁). The immunotherapeutic treatment is suitable for the treatment of primary tumors and/or for the treatment of recurring tumors.

The immunotherapeutic agent is preferably locally administered to the tumor site, i.e., to the site of a primary tumor before surgery or after surgery and optionally after chemotherapy. For the treatment of urothelial bladder carcinoma, the agent is preferably administered by vesicular instillation into the urinary bladder. For other tumors, the administration may involve local injection or, in case of lung tumors, pulmonal administration.

The immunotherapeutic agent of the invention may be administered as a first-line immunotherapy in patients, who have not been treated previously with an anti-tumor-immunotherapeutic agent such as standard BCG, or a follow-up immunotherapy in patients who have been treated previously with anti-tumor-immunotherapeutic agent such as standard BCG. The immunotherapeutic agent may be administered with a newly diagnosed solid tumor, e.g., a bladder carcinoma, or to patients, particularly patients with recurrent solid tumors, e.g., bladder carcinoma.

The immunotherapeutic agent is administered to the subject to be treated in an effective dose. For a human subject, the dose for an administration may be about 10⁶ to 10¹⁰ viable units (CFU), e.g. about 10⁷ to 10⁹ or 10⁸ to 10⁹ viable units. Preferably, the immunotherapeutic agent is administered several times, e.g. at least 3 times or at least 5 times up to 30 times, particularly about 15 times, at predetermined times during the treatment.

The immunotherapeutic agent is usually provided as a pharmaceutical preparation which comprises the recombinant Mycobacterial cell in solid form, e.g., a lyophilized or cryoconserved preparation, which is reconstituted with a suitable liquid carrier before use. Alternatively, the preparation may be provided in liquid form, e.g., as suspension.

In one embodiment, the immunotherapeutic agent of the invention is administered for the treatment of carcinoma in situ. A standard schedule may comprise weekly administration of the agent for at least 4, e.g., 4, 5, 6, 7 or 8 weeks as an induction therapy. The induction therapy should not start until 2-3 weeks after primary tumor surgery. After a treatment-free interval of, e.g., 4 weeks, administration may continue using maintenance therapy for at least 6 months or at least 1 year.

In a further embodiment, the immunotherapeutic agent is administered in an induction therapy in the prophylactic treatment of tumor recurrence. In this embodiment, therapy may start about 2-3 weeks after biopsy of the tumor site and be repeated, e.g., at weekly intervals for at least 4, e.g., 4, 5, 6, 7 or 8 weeks. In intermediate and high-risk tumors this may be followed by maintenance therapy.

Maintenance therapy may comprise long-term therapy, e.g., 6, 9 or 12 months therapy or even longer with treatments at monthly intervals. Alternatively, maintenance therapy may comprise 2, 3 or 4 administrations at weekly intervals, at month 3, 6, 12, 18, 24, 30 and 36.

In still a further embodiment, the immunotherapeutic agent, particularly recombinant BCG ΔUrec::Hly+, is used for the treatment of non-muscle invasive bladder cancer in patients with recurrence after standard BCG therapy. The immunotherapeutic agent is administered into the bladder according to a schedule involving weekly instillations during an induction phase with e.g. 6 weekly instillations, a first maintenance phase after about 3 months with e.g. 3 weekly instillations, a second maintenance phase after about 6 months with e.g. 3 instillations and a third maintenance phase after about 12 months with e.g. 3 instillations.

The administration as immunotherapeutic agent of the recombinant Mycobacterium cell to site of a solid tumor as described above, may be combined with further anti-tumor therapy, e.g., radiation and/or chemotherapy. Further, the immunotherapy as described above, may be combined with a non-tumor site specific administration of the recombinant Mycobacterium cell in order to provide a general stimulation of the immune system. This non-site specific administration may be effected as described in WO 2012/085101, e.g. before surgery of the primary tumor. In this case, the agent is preferably administered to a human subject in a dose of about 1-10×10⁵, preferably about 2-8×10⁵ cells. The agent is preferably administered as a single dose, e.g., by injection. Subcutaneous injection is preferred. Further it is preferred to administer the agent without adjuvant.

Further, the invention is described in more detail by the following Figures and Examples 1-3.

The immunotherapeutic agent “rBCG” used in these examples is recombinant M. bovis (BCG) Danish subtype Prague with an inactivated ureC sequence (ΔUrec) and without functional selection marker gene which expresses an Ag85B/Hly fusion protein as shown in SEQ ID No.2 (Hly+).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B—Immunohistochemistry for lymphocyte sub-typing in the urinary bladder tissue revealed the highest incidence of focal and multifocal lymphocytic infiltration with CD4 and CD8 positive cells in groups 2 and 3 treated once with 2X10⁶ or 2X10⁸ CFU rBCG/animal by intravesical instillation, whereas animals of groups 1 (control), 4 and 5 expressed CD4 and CD8 positive lymphocytes only as single cell infiltrate levels (diffuse infiltration). FIG. 1A shows focal and multifocal lymphocytic infiltration after administration of rBCG. FIG. 1B shows only diffuse and singular infiltration after administration of BCG medac (200X magnification).

FIG. 2A and FIG. 2B—Immunohistochemistry for lymphocyte sub-typing in the urinary bladder tissue revealed the highest incidence of multifocal lymphocytic infiltration with CD4 and CD8 positive cells in group 2 treated with 2X10⁶ CFU rBCG/animal by 6 intravesical instillations, followed by group 3 treated with 2X10⁸ CFU rBCG/animal by 6 intravesical instillations, whereas animals of groups 1 (control), 4 and 5 expressed CD4 and CD8 positive lymphocytes only as single cell infiltrate levels (diffuse infiltration). FIG. 2A shows focal and multifocal lymphocytic infiltration after administration of rBCG. FIG. 2B shows only diffuse and singular infiltration after administration of BCG medac (200X magnification).

EXAMPLE 1 SINGLE DOSE TOXICITY STUDY OF RECOMBINANT BCG (RBCG) IN RATS FOLLOWING INTRAVESICULAR INSTILLATION

1.1 Conduct of Study

Test item rBCG lyophilized (rBCG Danish subtype Prague ΔUrec::Hly + w/o functional selection marker gene) Approximate viable 5.41 × 10⁸ CFU/vial counts Reference item BCG medac Approximate viable counts 2 × 10⁸ to 2 × 10⁹ CFU/vial Test species/Strain/Stock Rat/CD ®/Crl:CD(SD) Breeder Charles River Laboratories, Research Models, and Services, Germany GmbH Sandhofer Weg 7 97633 Sulzfeld, Germany Number and sex of 23 female animals; animals 3 animals for group 1; 5 animals for groups 2 to 5. Dose regime Group 1: Control (diluent) Group 2: ~2 × 10⁶ CFU rBCG (lyophilized)/animal Group 3: ~2 × 10⁸ CFU rBCG (lyophilized)/animal Group 4: ~2 × 10⁶ CFU rBCG (frozen w/o cryoprotectant)/ animal Group 5: ~2 × 10⁶ CFU BCG medac/animal Route of administration Intravesical instillation in the bladder Frequency of administration Single dose on test day 1. Administration volume 500 μL/animal Duration of study 12 adaptation days 4 in-life test weeks 28 incubation days

1.2 Results

Mortality None of the animals died prematurely. Clinical signs No changes of behaviour, external appearance or condition of faeces were observed for any animal at any treatment. Body weight The body weight of all animals of all dose groups was in the normal range throughout the course of the study. Food and drinking The food intake of all animals of all dose water consumption groups was in the normal range throughout the course of the study. The visual appraisal of the drinking water consumption did not reveal any test or reference item-related influence. IL-2 levels The IL-2 levels in urine and serum of all animals of all groups were below the lower limit of quantification. Macroscopic post mortem No test or reference item-related changes findings were noted. Organ weights No test or reference item-related changes were noted. CFU counts rBCG (groups 2 to 4) vs. control (group 1) No test-item related CFU counts were noted for the examined organs and the blood of the animals treated once with an intravesical instillation of 2 × 10⁶ or 2 × 10⁸ CFU rBCG (lyophilized)/animal, or of 2 × 10⁶ CFU rBCG (frozen)/animal. In particular, no CFU counts at all were noted for the urinary bladder four weeks after instillation of the test item, indicating a rapid clearance of the administered mycobacteria from the site of instillation. No differences were noted between the animals treated with 2 × 10⁶ or 2 × 10⁸ CFU rBCG (lyophilized)/animal or with 2 × 10⁶ CFU rBCG (frozen)/animal and the control animals. BCG medac (group 5) vs. control (group 1) No reference-item related CFU counts were noted for the examined organs and the blood of the animals treated once with an intravesical instillation of 2 × 10⁶ BCG medac/animal. In particular, no CFU counts at all were noted for the urinary bladder four weeks after instillation of the reference item, indicating a rapid clearance of the administered mycobacteria from the site of instillation. No differences were noted between the animals treated with 2 × 10⁶ BCG medac/animal and the control animals. rBCG (groups 2 to 4) vs. BCG medac (group 5) No difference in CFU counts was noted for the examined organs and the blood of the animals treated once with an intravesical instillation of 2 × 10⁶ or 2 × 10⁸ CFU rBCG (lyophilized)/animal, or of 2 × 10⁶ CFU rBCG (frozen)/animal compared to the reference group treated in the same way with 2 × 10⁶ CFU BCG medac/animal. Immunohistochemistry Immunohistochemistry for lymphocyte sub- typing in the urinary bladder tissue revealed the highest incidence of focal and multifocal lymphocytic infiltration with CD4 and CD8 positive cells in groups 2 and 3 treated once with 2 × 10⁶ or 2 × 10⁸ CFU rBCG (lyophilized)/animal by intravesical instillation, whereas animals of groups 1 (control), 4 and 5 expressed CD4 and CD8 positive lymphocytes only as single cell infiltrate levels (diffuse infiltration). Nearly all animals (12 of 13) of groups 1 (control), 4 and 5 expressed CD4 and CD8 positive lymphocytes only as single cell infiltrate levels (diffuse infiltration). In contrast nearly all animals (9 of 10) of groups 2 and 3 contained CD4 and CD8 positive lymphocytes in focal or multifocal lymphocyte infiltrates in addition to the diffuse infiltration. Neutrophilic granulocytes could rarely be detected in any slides examined.

In conclusion, no signs of toxicity were noted for the animals treated with the test item, the reference item or the dilutent. There were no differences in the systemic spread of mycobacteria in blood and organs between rBCG and BCG medac for the treatment by intravesical instillation. No test-item related CFU counts were noted for the examined organs and the blood of the animals treated once with an intravesical instillation of rBCG compared to the control animals. In particular, no CFU counts at all were noted for the urinary bladder 4 weeks after instillation of the test item, indicating a rapid clearance of the administered mycobacteria from the site of instillation.

Immunohistochemistry for lymphocyte sub-typing in the urinary bladder tissue revealed the highest incidence of focal and multifocal lymphocytic infiltration with CD4 and CD8 positive cells in groups 2 and 3 treated once with 2×10⁶ or 2×10⁸ CFU rBCG/animal by intravesical instillation, whereas animals of groups 1 (control), 4 and 5 expressed CD4 and CD8 positive lymphocytes only as single cell infiltrate levels (diffuse infiltration). Nearly all animals (12 of 13) of groups 1 (control), 4 and 5 expressed CD4 and CD8 positive lymphocytes only as single cell infiltrate levels (diffuse infiltration). In contrast, nearly all animals (9 of 10) of groups 2 and 3 contained CD4 and CD8 positive lymphocytes in focal or multifocal lymphocyte infiltrates in addition to the diffuse infiltration. FIG. 1A shows focal and multifocal lymphocytic infiltration after administration of rBCG. FIG. 1B shows only diffuse and singular infiltration after administration of BCG medac (200× magnification).

EXAMPLE 2 REPEATED DOSE TOXICITY STUDY OF RECOMBINANT BCG (RBCG) IN RATS FOLLOWING INTRAVESICULAR INSTILLATION

2.1 Conduct of Study

Test item rBCG lyophilized (rBCG Danish subtype Prague ΔUrec::Hly + w/o functional selection marker gene) Approximate viable counts 5.41 × 10⁸ CFU/vial Reference item BCG medac Approximate viable counts 2 × 10⁸ to 2 × 10⁹ CFU/vial Test species/Strain/Stock Rat/CD ®/Crl:CD(SD) Breeder Charles River Laboratories, Research Models, and Services, Germany GmbH Sandhofer Weg 7 97633 Sulzfeld, Germany Number and sex of animals 23 female animals; 3 animals for group 1; 5 animals for groups 2 to 5. Dose regime Group 1: Control (diluent) Group 2: ~2 × 10⁶ CFU rBCG (lyophilized)/animal Group 3: ~2 × 10⁸ CFU rBCG (lyophilized)/animal Group 4: ~2 × 10⁶ CFU rBCG (frozen w/o cryoprotectant)/ animal Group 5: ~2 × 10⁶ CFU BCG medac/ animal Route of administration Intravesical instillation in the bladder Frequency of administration Repeated administration; once weekly on test days 1, 8, 15, 22, 29, and 36. Administration volume 500 μL/animal Duration of study 21 adaptaion days 9 in-life test weeks 28 incubation days

2.2 Results

Mortality None of the animals died prematurely. Clinical signs No changes of behaviour, external appearance or condition of faeces were observed for any animal at any treatment. Body weight The body weight of all animals of all dose groups was in the normal range throughout the course of the study. Food and drinking The food intake of all animals of all dose water consumption groups was in the normal range throughout the course of the study. The visual appraisal of the drinking water consumption did not reveal any test or reference item-related influence. IL-2 levels The IL-2 levels in urine and serum of all animals of all groups were below the lower limit of quantification. Delayed type hyper- No delayed type hypersensitivity was noted. sensitivity (DTH assay) Macroscopic post mortem No test or reference item-related changes findings were noted. Organ weights No test or reference item-related changes were noted. CFU counts rBCG (groups 2 to 4) vs. control (group 1) No test-item related CFU counts were noted for the examined organs and the blood of the animals treated six times with an intravesical instillation of 2 × 10⁶ or 2 × 10⁸ CFU rBCG (lyophilized)/animal, or of 2 × 10⁶ CFU rBCG (frozen/w/o cryoprotectant)/animal. In particular, no CFU counts at all were noted for the urinary bladder four weeks after the last instillation of the test item, indicating a rapid clearance of the administered mycobacteria from the site of instillation. No differences were noted between the animals treated with 2 × 10⁶ or 2 × 10⁸ CFU rBCG (lyophilized)/animal or with 2 × 10⁶ CFU rBCG (frozen)/animal and the control animals. BCG medac (group 5) vs. control (group 1) No reference-item related CFU counts were noted for the examined organs and the blood of the animals treated six times with an intravesical instillation of 2 × 10⁶ BCG medac/animal. In particular, no CFU counts at all were noted for the urinary bladder four weeks after the last instillation of the reference item, indicating a rapid clearance of the administered mycobacteria from the site of instillation. No differences were noted between the animals treated with 2 × 10⁶ BCG medac/animal and the control animals. rBCG (groups 2 to 4) vs. BCG medac (group 5) No difference in CFU counts was noted for the examined organs and the blood of the animals treated six times with an intravesical instillation of 2 × 10⁶ or 2 × 10⁸ CFU rBCG (lyophilized)/ animal, or of 2 × 10⁶ CFU rBCG (frozen)/animal compared to the reference group treated in the same way with 2 × 10⁶ CFU BCG medac/animal. Immunohistochemistry Immunohistochemistry for lymphocyte sub- typing in the urinary bladder tissue revealed the highest incidence of multifocal lymphocytic infiltration with CD4 and CD8 positive cells in group 2 treated with 2 × 10⁶ CFU rBCG/animal by 6 intravesical instillations, followed by group 3 treated with 2 × 10⁸ CFU rBCG/animal by 6 intravesical instillations, whereas animals of groups 1 (control), 4 and 5 expressed CD4 and CD8 positive lymphocytes only as single cell infiltrate levels (diffuse infiltration). Neutrophilic granulocytes could rarely be detected in any slides examined.

In conclusion, no signs of toxicity were noted for the animals treated with the test item, the reference item or the diluent. There were no differences in the systemic spread of mycobacteria in blood and organs between rBCG and BCG medac for the treatment by intravesical instillation. No test-item related CFU counts were noted for the examined organs and the blood of the animals treated six times with an intravesical instillation of rBCG compared to the control animals. In particular, no CFU counts at all were noted for the urinary bladder 4 weeks after the last instillation of the test item, indicating a rapid clearance of the administered mycobacteria from the site of instillation.

Immunohistochemistry for lymphocyte sub-typing in the urinary bladder tissue revealed the highest incidence of multifocal lymphocytic infiltration with CD4 and CD8 positive cells in group 2 treated with 2×10⁶ CFU rBCG/animal by 6 intravesical instillations, followed by group 3 treated with 2×10⁸ CFU rBCG/animal by 6 intravesical instillations, whereas animals of groups 1 (control), 4 and 5 expressed CD4 and CD8 positive lymphocytes only as single cell infiltrate levels (diffuse infiltration). FIG. 2A shows focal and multifocal lymphocytic infiltration after administration of rBCG. FIG. 2B shows only diffuse and singular infiltration after administration of BCG medac (200× magnification).

EXAMPLE 3 PHASE I/II OPEN LABEL CLINICAL TRIAL ASSESSING SAFETY AND EFFICACY OF INTRAVESICAL INSTILLATION OF RECOMBINANT BCG (RBCG) IN HUMAN PATIENTS WITH RECURRENT NON-MUSCLE INVASIVE BLADDER CANCER AFTER STANDARD BCG THERAPY

3.1 Clinical Protocol

The recombinant BCG (as defined above) is currently applied in clinics as part of a Phase I/II clinical trial by instillation into bladder. The clinical trial phase I/II aims at assessing safety and efficacy of intravesical instillation of rBCG in human patients with recurrent non-muscle invasive bladder cancer after standard BCG therapy. rBCG is administered into bladder in 15 weekly instillations (induction phase: instillation 1-6, maintenance 3 months: instillation 7-9, maintenance 6 months: instillation 10-12, maintenance 12 months: instillation 13-15).

The primary endpoint of the phase I is dose limiting toxicity (DLT) of intravesical rBCG instillations in patients with recurrence after standard BCG therapy in non-muscle invasive bladder cancer. The DLT period corresponds to 3 instillations plus 1 week and covers acute toxicities induced by treatment. Patients are treated in two cohorts of three, following the rules of a 3+3 design (dose de-escalation rules: if patients treated at dose level 1 show signs of DLT, dose of instilled rBCG will be reduced to level −1, which is 10 times lower than level 1).

The dose levels are as follows:

-   -   Dose level 1: 1−19.2×10⁸ CFUs of rBCG     -   Dose level −1: 1−19.2×10⁷ CFUs of rBCG

3.2 Current Status

The clinical trial currently is recruiting patients in trial sites in Switzerland (Basel, Geneva, Chur, Bern, Bellinzona, St. Gallen) within the frame of the Phase I.

The DLT period for the first three patients (cohort 1) was completed on 23 Feb. 2016. Safety data, including DLT data, from this cohort has been collected by the sponsor and delivered to the Independent Safety Data Committee (ISDC). Since no DLT has been observed, the next three patients (second cohort) were allowed to be enrolled simultaneously and the recombinant BCG dose was maintained at dose level 1. An approval from the ISDC was received and all parties involved in the trial have been informed about this on 9 Mar. 2016 by the sponsor. In addition, no tumor recurrence has been observed in this cohort of patients at the time of evaluation.

Currently recruitment of the second cohort is ongoing, with the first patient already enrolled in this cohort. 

The invention claimed is:
 1. A method for the immunotherapy of bladder carcinoma in a human subject, comprising a) obtaining a urease C-deficient recombinant Mycobacterium (M.) bovis Bacillus Calmette-Guérin (BCG) cell from strain Danish subtype Prague as an immunotherapeutic agent comprising a recombinant nucleic acid molecule encoding a fusion polypeptide comprising: (i) a domain capable of eliciting an immune response, and (ii) a Listeria phagolysosomal escape domain listeriolysin (Hly), wherein said urease C-deficient recombinant Mycobacterium (M.) bovis Bacillus Calmette-Guérin (BCG) cell is provided in lyophilized form, b) reconstituting said urease C-deficient recombinant Mycobacterium (M.) bovis Bacillus Calmette-Guérin (BCG) cell in a liquid carrier, and c) administering said reconstituted urease C-deficient recombinant Mycobacterium (M.) bovis Bacillus Calmette-Guérin (BCG) cell by vesicular instillation into a subject's urinary bladder.
 2. The method of claim 1, wherein the domain capable of eliciting an immune response is selected from immunogenic peptides or polypeptides from M. bovis or M. tuberculosis.
 3. The method of claim 1, wherein the recombinant nucleic acid molecule does not comprise any functional selection marker.
 4. The method of claim 1, wherein the fusion polypeptide comprises (a) a domain capable of eliciting an immune response comprising the amino acid sequence from aa.41 to aa.51 in SEQ ID NO:2, and (b) a Listeria phagolysosomal escape domain encoded by a nucleic acid molecule selected from (i) a nucleotide sequence comprising nucleotides 211-1722 as shown in SEQ ID No.1, (ii) a nucleotide sequence which encodes the same amino acid sequence as the sequence from (i), and (iii) a nucleotide sequence hybridizing under stringent conditions with a full length complement of the sequence from (i) or (ii), wherein said stringent conditions comprise washing for one hour with 0.2× or 1× SSC and 0.1% SDS at a temperature of 55-68° C.
 5. The method of claim 1, wherein the bladder carcinoma is selected from the group consisting of non-invasive bladder carcinoma, non-invasive papillary carcinoma (Ta), and a tumor invading subepithelial connective tissue (T1).
 6. The method of claim 5, wherein said non-invasive bladder carcinoma is carcinoma in situ (Tcis).
 7. The method of claim 1, wherein the immunotherapeutic agent is administered after surgery.
 8. The method of claim 1 wherein said human subject is a patient with newly diagnosed or recurrent bladder carcinoma who has not been treated previously with standard BCG.
 9. The method of claim 1 wherein said human subject is a patient with recurrent bladder carcinoma who has been treated previously with standard BCG.
 10. The method of claim 1, wherein the immunotherapeutic agent is administered into the human subject's urinary bladder according to a schedule involving weekly instillations during an induction phase, a first maintenance phase after about 3 months, a second maintenance phase after about 6 months and a third maintenance phase after about 12 months.
 11. The method of claim 10, wherein the immunotherapeutic agent is administered into the human subject's urinary bladder according to a schedule involving weekly instillations during an induction phase with 6 weekly instillations, a first maintenance phase after about 3 months with 3 weekly instillations, a second maintenance phase after about 6 months with 3 instillations and a third maintenance phase after about 12 months with 3 instillations.
 12. The method of claim 1, wherein the immunotherapeutic agent is administered at a dose of from about 10⁶ to 10¹⁰ CFU per administration.
 13. The method of claim 1, wherein the immunotherapy is combined with a non-tumor site specific administration of the recombinant M. bovis cell.
 14. The method of claim 1 wherein focal and/or multifocal lymphocytic infiltration at the site of administration is obtained.
 15. The method of claim 14 wherein focal and/or multifocal tissue infiltration with CD4 and CD8 T cells is obtained.
 16. The method of claim 4, wherein said stringent conditions comprise washing for one hour with 0.2×SSC and 0.1% SDS at a temperature of 55-68° C.
 17. A method for the immunotherapy of bladder carcinoma in a human subject, comprising administering to said subject a urease C-deficient recombinant Mycobacterium (M.) bovis Bacillus Calmette-Guérin (BCG) cell from strain Danish subtype Prague as an immunotherapeutic agent comprising a recombinant nucleic acid molecule encoding a fusion polypeptide comprising: (a) a domain capable of eliciting an immune response, and (b) a Listeria phagolysosomal escape domain listeriolysin (Hly), wherein said immunotherapeutic agent is provided in lyophilized form and reconstituted with a liquid carrier prior to administration by vesicular instillation into the urinary bladder wherein the immunotherapeutic agent is administered into the human subject's urinary bladder according to a schedule involving weekly instillations during an induction phase, a first maintenance phase after about 3 months, a second maintenance phase after about 6 months and a third maintenance phase after about 12 months. 